1. Field of the Invention
The present invention is broadly concerned with multiple-peptide channel assemblies which provide transport of anions through epithelial cell membranes wherein the preferred peptides have from about 16-31 amino acid residues and are soluble in water to a level of at least 5 mM; such channel assemblies can be used in the treatment of diseases such as cystic fibrosis (CF) and adult polycystic kidney disease (APKD). More particularly, the invention pertains to such channel assembly forming peptides, and corresponding methods of use, wherein the peptides are derived from a segment of a native (i.e., naturally occurring) channel protein and have their water solubilities enhanced by modification of the C- or N-ends thereof modified with a plurality of polar amino acid residues such as lysine. The polar amino acids (both DNA coding and non-coding), which are not limited to the all L-stereoconfiguration, include: lysine, arginine, glutamic acid, aspartic acid, diaminopropionic acid, diaminobutyric acid, ornithine, and homolysine. These amino acids are characterized by their ability to adopt different charged states at different pH values. Under physiological conditions, pH 7.2-7.4, the side chain amino (+) or carboxylic acids (−) are in the charged or ionized state. Still more particularly, the invention pertains to derivatives of the M2GlyR sequence, which remain predominantly in monomer form when in solution, have a desired amount of helical configuration, and alter the transepithelial electrical resistance of cell layers to a greater extent than was heretofore possible. Additionally, one aspect of the invention pertains to derivatives of the M2GlyR sequence that modulate the permeability to polar and non-polar solutes across tight junctions that join epithelial cells into a confluent, electrically resistive layer.
2. Description of the Prior Art
Introduction. A major problem in CF is the inability of airway epithelia to secrete fluid. The resulting changes in the composition of the mucous coating the airway epithelia result in infection and subsequent inflammation, scarring, and eventual pulmonary destruction. The basis of the problem is the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane of the epithelial cells. This leads to an increase in the absorption of salt and water and an inability to respond to appropriate stimuli by secreting chloride and water. CFTR is an anion channel; in addition it down-regulates sodium channel expression or function and modulates the activity of other ion permeation pathways (e.g., an outwardly rectifying chloride channel (ORCC) and some potassium channels). These properties of CFTR enable the airway cells to secrete chloride and this drives the secretion of sodium and water.
A synthetic-23-residue α-helical peptide designated M2GlyR forms anion-selective channels in phospholipid bilayers. This peptide has the amino acid sequence of the putative transmembrane segment, typically designated M2, of the strychnine-binding α subunit of the glycine receptor.
The origin and properties of M2GlyR. The glycine receptor is a membrane protein present in post-synaptic membranes. Binding of glycine activates a Cl− conducting channel, leading to hyperpolarization of the membrane and inhibition of the synapse. The receptor consists of two major glyco-polypeptides, an α subunit of 48 kd and a β subunit of 58 kd, and a receptor-associated cytoplasmic protein of 93 kd. Strychnine, an antagonist of the glycine receptor, binds only to the α subunit. Messenger RNA corresponding to this subunit leads to the expression of functional, glycine-activated, Cl− channels upon injection into Xenopus oocytes.
The glycine receptor channel in cultures of embryonic mouse spinal cord is selective for monovalent anions, with conductances of 27 and 46 pS in 145 mM Cl− solution. Pharmacological studies suggested the presence of two sequentially occupied anion binding sites in the channel. These sites are considered to be the functional correlates of the positively charged amino acids bordering the M2 segment of the α subunits. This finding led to the development of the synthetic peptide with the sequence of the M2 segment of the glycine receptor.
Electrical recordings from phospholipid bilayers containing M2GlyR showed single-channel conductances of 25 pS and 49 pS in symmetric 0.5 M KCl with channel open lifetimes in the millisecond range. Single channel events occurred in 0.5 M N-methyl-D-glucamine Cl but not in sodium gluconate, indicating that the channel is anion selective. A transference number for anions of 0.85 was calculated from reversal potential measurements under a 5-fold KCl concentration gradient.
After insertion into the lipid bilayers the monomeric peptides self-assemble to form oligomers that exhibit various amplitudes of ion conductance. To gain control over the aggregate number of monomers that form a functional ion-selective channel, four identical M2GlyR peptide units were tethered to a 9-amino acid carrier template to form a four-helix bundle protein. This tetramer, self-inserted into lipid bilayers, and formed uniform 25 pS channels. The 49 pS conductance described above is presumed to be due to the presence of a pentamer.
The tetrameric channel was blocked by the Cl− channel blockers 9-anthracene carboxylic acid (9-AC) and niflumic acid (NFA). It was not blocked by QX-222, an analogue of lidocaine and a blocker of cation-selective channels. Strychnine, an antagonist of the glycine receptor, does not block the channel-forming tetramer. Strychnine is presumed to bind to the ligand-binding domain of the receptor exposed to the extracellular surface but not to the channel domain.
Structure of channel forming peptides. While great strides have been made in the area of channel function and regulation, using the intact protein or in some cases purified channel proteins reconstituted into model membranes, many aspects of channel function remain unresolved. A K+ channel from streptomyces lividans was crystallized and the structure determined at 3.2 Angstroms. This structure has served as a model for other ion channels using homology modeling methodologies. This structure, however, is for a 4 subunit channel as opposed to the five subunit channel proposed for the glycine receptor.
Considerable structural data exist for the related class of channel forming peptides (CFPs). Naturally occurring CFPs constitute a class of bioactive peptides. In the present application, the claimed CFPs are peptides that form discrete ion-selective conducting pores rather than sequences such as Magainins which form large non-selective holes in the membrane. These CFPs have channels that are much smaller in size and contain only a ring of short peptide chains organized around the central ion conducting pore in the lipid bilayer. These channels are unique in that they assemble by the oligomerization of a single peptide. These structures are models for studying the structure and function of the various regulated channels that occur in nature. This class of CFPs includes: the α-aminoisobutyric acid-containing channels such as alamethicin and zervamicin, and a number of toxins and venoms such as melittin, cecropins, mast cell degranulating peptides, and the defensins. Melittin is somewhat of a special case because it forms channels only at low concentrations; at higher concentrations it acts as a lytic agent. In some cases CFPs assemble spontaneously upon insertion into the bilayer while in the remaining cases the assembly requires an electrical potential across the membrane (Vm).
The structure of the channels arising from the assembly of these peptides vary from trimers to hexadecamers associated in the form of helical bundles or β-barrels. The most widely accepted model that is in accord with the model for channel proteins has the helices arranged with their dipoles all pointing in the same direction (parallel). Since CFP channels, unlike authentic channel proteins, are not generated from the association of large protein subunits, alternative stabilization schemes must be invoked to account for the presence of this higher energy arrangement of parallel segments. These could include aligning the dipoles in response to the presence of the membrane potential and/or an increase in the favorable inter-molecular interactions promoted by the parallel assembly. Most CFPs form multiple size bundles of parallel segments (e.g., n=4, 5, 6) that can spontaneously increase or decrease in size upon the addition or deletion of a peptide monomer to or from the channel assembly. These observations imply that enough information is contained in a single channel forming polypeptide to drive the correct folding, assembly, and activity of these channels.
The activity of these assembled molecules, the opening and closing of the channels on the millisecond time scale, has been ascribed to numerous effects. Three different helical motions have been implicated: the bending and twisting of the helices, rigid-body fluctuations of the entire assembled structure with the lipid bilayer, and rotational motions of the polypeptide around its helical axis. Another hypothesis suggests that channel activity is a consequence of a conformational change that is transmitted along the helical axis. Others suggest that the movement of individual amino acid side-chains could provide this function, and one group contends that an electron transfer could disrupt a hydrogen bonding of four tyrosines in K+ channels.
Fluorescence, Fourier transform infrared spectroscopy (FTIR), and circular dichroism (CD) measured in organic solvents, phospholipid micelles, liposomes, or oriented phospholipid bilayers, have been successfully used to probe the solution and membrane-bound conformations of these CFPs. Computer modeling studies have been performed to estimate the energetics of moving an ion across a lipid bilayer through a pore generated by a bundle of transmembrane helices. Structural experiments using NMR are yielding important results. In general, these studies have provided several conclusions concerning the solution behavior and membrane interactions of CFPs. Amphipathic helical peptides can co-exist as monomers and aggregates in solution. Monomers interact much more readily with lipid bilayers and micelles. Depending on the peptide to lipid ratio, type of lipid, ionic strength, solution pH, and lipid hydration, the peptide will preferentially orient itself either parallel to or perpendicular to the plane of the bilayer. Many CFPs do not require a potential difference across the bilayer to insert spontaneously into the bilayer. Once in the membrane, the helices associate in a time- and concentration-dependent manner to form the multistate helical bundles. It is these assemblies that conduct ions across the bilayer. These studies, when considered together, reveal the transmembrane amphipathic helix to be a dynamic structure. The ability to oligomerize in the membrane into stable ring structures, with a central aqueous pore capable of opening and closing, appears to be driven by the asymmetrical alignment of hydrophilic and hydrophobic amino acid residues that seem to obey a unique set of rules.
Putative channel forming segments from large channel proteins behave much like the small naturally occurring CFPs described above. They spontaneously insert into bilayers and self-assemble into an ion-conducting structure, presumably comprised of a parallel array of α-helices. These structures retain biological activities reminiscent of the associated native proteins. These channel-forming structures are reasonable models for exploring both the oligomerization of transmembrane segments and for defining the molecular events that give rise to channel activity. The beauty of this system emanates from the appearance of a measurable activity (i.e., ion permeation) that arises from the assembly of an amphipathic transmembrane helix. The activity allows measurement of the effects of amino acid substitutions on either the size of the assemblies or the contribution of the residues to ion selectivity or translocation. The number of helices per channel can be precisely controlled, thus preventing multiple oligomerization states, by tethering the helical segments to a peptide backbone during synthesis. The small size of these assemblies makes them ideally suited for NMR structural studies using either detergent micelle solution NMR or oriented bilayer solid-state NMR.
Pharmacological studies have been a relatively recent addition to the single channel analysis of these model CFP channels. Using a four helix bundle CFP derived from the human L-type dihydropyridine sensitive Ca2+ channel, the binding of a local anaesthetic as well as a number of calcium channel blockers with binding affinities on the order of those observed for the full length calcium channel protein have been observed. This avenue of investigation adds a sensitive method of discriminating between channels that truly mimic their parent structures as opposed to those that might produce non-discriminating ionic pores. Once the three dimensional structure for one of the synthetic channels has been solved, rational drug design of both channel agonists and antagonists may be attempted using these coordinates.
Membrane proteins are generally acknowledged to be the most difficult class of proteins for detailed structural analysis. The studies presented above clearly demonstrate the utility of working with small synthetic CFPs, as model systems, to study events involved in peptide association with lipid membranes, insertion into membranes, and assembly into ion-conducting oligomers. The amphipathic helix is a suitable structural motif for the pore of channel proteins that also contributes to the organization, size, function, and stabilization of ionic channels. As an assembled structure, these helical bundles can be used to investigate the structure, organization, and function of channels.
Application of synthetic peptides to biological membranes. Extensive evidence indicates that Cl− secretion drives fluid secretion across Madin-Darby canine kidney (MDCK) cells, across cells cultured from the cystic epithelium of the kidneys of patients with autosomal dominant polycystic kidney disease (APKD), and that a Cl− channel is involved in fluid secretion. Indeed there is now extensive data indicating that CFTR is the channel involved in that secretion by APKD cells. Apparently, a net secretion of Cl− into the lumen of the cysts leads to an increase in water volume in the cysts, ultimately resulting in kidney dysfunction. However, although there is a precedent for the application of synthetic channel-forming peptides to cells, no one previously has used channel-forming peptides to treat symptoms of any disease.
Tight junctions. Epithelial and endothelial cells form monolayers within the body that generate and separate fluid compartments of distinct compositions and protect the interstitial space from environmental factors. These activities are highly desirable in that they allow for physiological function and, in general, are associated with bodily health. However, in numerous pathological states the epithelium or endothelium provides a barrier that precludes therapeutic access to the targeted site. Notably, the intestine is a barrier to drug absorption, the nephron is a barrier to drug retention, and brain vessel endothelium inhibits access of psychoactive and other therapeutic drugs to the brain. Thus, modulation of the epithelial or endothelial barrier function is key to delivering therapy in many life-threatening situations.
The barrier function of epithelial cells is performed by tight junctions: complex, highly regulated, protean structures. The multitude of ‘junctional’ and associated proteins that participate in the barrier function suggests that one or more of the components might be targeted for therapeutic interventions. Transient openings of these junctions are required for a variety of bodily functions including sperm maturation, extravasation of lymphocytes across endothelia and nutrient uptake associated with activity of the Na+/glucose transporter. Pathology associated with aberrant function and dysregulation of the tight junction includes cancer metastases, autoimmune dysfunction, coeliac disease, and inflammatory bowel disease. Tight junctions are targets of bacterial toxins such as the Vibrio cholerae zonula occludens toxin (ZOT) and Clostridium difficile toxins TcdA and TcdB. Tight junction permeability is tightly regulated (see FIG. 31, adapted from Mitic, L. L., C. M. Van Itallie, and J. M. Anderson. Molecular physiology and pathophysiology of tight junctions I. Tight junction structure and function: lessons from mutant animals and proteins. Am J Physiol Gastrointest Liver Physiol 279: G250-4, 2000). The cytoskeleton of actin microfilaments, associated with myosin and other cellular proteins, maintains the morphology of epithelial cells. An intracellular ring of actin and myosin at the apical/lateral interface (the perijunctional actomyosin ring) provides a scaffold for the tight junctions between epithelial or endothelial cells. The primary transmembrane structural components of tight junctions are the claudin family proteins, junctional adhesion molecule (JAM) and occluden. These proteins interact directly with the ZO family proteins, which link them to the perijunctional ring of the cytoskeleton. These proteins also interact with several regulatory/signaling molecules. The ZO proteins contain a guanylate kinase (GUK) domain as well as a src homology 3 (SH3) domain and a PDZ domain. The atypical PKC isotype specific interacting protein (ASIP) and the ras binding protein AF-6 also contain PDZ domains, and have been shown to associate with junctional complexes. PKC phosphorylates occluden, which results in its translocation to the tight junction. In subconfluent epithelial cell cultures, ZO-1 localizes to the nucleus but is located at the junctions in confluent cultures of epithelial cells. Myosin light chain kinase phosphorylation of the myosin II (regulatory subunit) is associated with contraction of the perijunctional ring and increases in paracellular permeability. Protein kinase A (PKA) activation increases conductance, but not permeability to large molecules across tight junction, while activation of PKC increases paracellular permeability. Barrier function of the tight junction is also affected by calcium levels, which may be under the control of PKC. Rho GTPase family members control organization of the actin cytoskeleton, (specifically cdc42). Rab GTPase proteins, which play a regulatory role in vesicular trafficking, such as rab13 and rab3b, appear to play a role in junctional regulation that remains undefined. These observations demonstrate that numerous cellular components might be targeted to modulate the paracellular conductance.
Endothelial tight junctions share many components with epithelial tight junctions although distinct extracellular modulators impinge on their function. Inflammatory agents can increase endothelial permeability; these mechanisms include bradykinin, which increases blood-brain barrier permeability by acting on B2 receptors, serotonin, which shows evidence for activation of 5-HT2 receptors and a calcium-dependent permeability increase, and histamine, which is mediated by H2 receptors and elevation of [Ca2+]i and an H1 receptor-mediated reduction in permeability coupled to an elevation of cAMP. Mechanisms induced by ionmycin have been shown to increase albumin clearance and decrease electrical resistance across bovine pulmonary microvascular and macrovascular endothelial cell monolayers. The ionmycin seems to produce barrier dysfunction by mechanisms that are independent of myosin light chain kinase activation and reductions in endothelial cell tethering forces via inhibition of protein kinase A and tyrosine kinase activities. In addition to these studies, many reports show that low molecular weight compounds may penetrate endothelial monolayers. Findings reveal that opening of the blood brain barrier by arachidonic acid, mediated by granulocytes and/or their products, can be attributed to the acid opening the blood-brain barrier for small molecular weight compounds at concentrations of 30-300 μM and 3 mM for larger molecular weight compounds. In other words, arachidonic acid, generated in response to granulocytes and their products, modulates the endothelial barrier to allow permeation by small solutes at low concentration (<300 μM) and larger solutes at higher concentrations. Recent studies show that intra-arterial administration of alkylglycerol represents a well controllable method for enhanced drug delivery to the brain and to brain tumors through the blood brain barrier; in the presence of alkylglycerols at concentrations of 10-30 mM, a reversible and concentration-dependent enrichment of administered drugs was observed. Another experiment revealed that leucine enkephalin enhanced bovine brain microvessel endothelial cell monolayer permeability either by altering paracellular openings or through formation of a small pore in the monolayers to allow preferential penetration of low molecular weight or small molecular size substances. These observations highlight that ongoing regulation of endothelial tight junctions occurs in vivo. The results further suggest that there is not a well-targeted pharmaceutical on the market that can be used to modulate the endothelial barrier.
Numerous techniques are currently being evaluated for the ability to selectively and transiently modulate epithelial and endothelial barrier function. In addition to the methods listed above, pharmaceuticals are being linked to actively transported peptides, as a means to cross the blood-brain barrier. While this allows very selective targeting, the method requires a unique synthetic process for every transported compound. Alternatively, methods are being developed to reduce epithelial tight junctions enough to allow large molecules to diffuse to the interstitial space. Both calcium chelators and surfactants have been employed, but have unacceptable side effects including global changes in cell function and diminished cell adhesion. Alternatively, the zonula occludens toxin of Vibrio cholerae (ZOT) provides a naturally occurring alternative for increasing the permeability of small intestine epithelia. ZOT and its eukaryotic homologue, zonulin, interact with an epithelial membrane receptor that leads to a reduction in epithelial electrical resistance, presumably by activation of PKCα. The effects of ZOT are rapid in onset (<20 minutes) and readily reversible on washout. Thus ZOT is an excellent candidate as an adjunct to standard therapy to increase oral bioavailablility of large molecules across intestinal epithelium. In fact, ZOT has been used to increase the permeation of anticonvulsant drugs across epithelial monolayers, to increases the uptake of PEG 4000 from rabbit small intestine and into the bloodstream. In diabetic rats, the bioavailability of oral insulin was sufficient to control blood glucose to the same degree as parenteral administration. However, ZOT has some drawbacks as a more generalized therapeutic in that it is a large peptide (399 a.a.) and has a relatively small therapeutic target. Effects are observed only in the small intestine where distinct receptors are present. It was subsequently reported that an 8 a.a. peptide could fully inhibit the effects of ZOT on small intestine. Thus, the small peptides that we are developing may have some therapeutic advantages in that a wider tissue applicability may be observed (effect on renal, reproductive, intestinal, and airway epithelia have been observed) and it is a small peptide making it much more economical to commercially produce.
Channel forming peptides have not previously been used to modulate or regulate tight junctions.
U.S. Pat. No. 5,543,399 describes the purification and lipid reconstitution of CFTR protein and CF therapy making use of that protein. There is no teaching or suggestion in this reference of the use of relatively small, easily prepared pure peptides, and particularly peptides which are fragments of channel-forming proteins.
U.S. Pat. No. 5,368,712 teaches the use of small peptides reconstituted in artificial membranes as diagnostic tools. This patent does not describe any therapeutic applications using such peptides.
U.S. Pat. No. 6,077,826, the content of which is hereby incorporated by reference, describes the use of multiple-peptide channel assemblies which transport anions through epithelial cells, synthetic peptides capable of forming such assemblies, channel assemblies which alter the flux of water across these cells, and channel assemblies which alter the transepithelial electrical resistance of cells. These assemblies were based on the M2GlyR sequence and were modified to increase their solubility. However, the activity of these assemblies is limited to about 15 μA/cm2 at a concentration of about 500 μM. Additionally, the peptides of this invention form multimers in solution, which have decreased affinity for membranes and suffer from solution aggregation.
Accordingly, what is needed in the art are channel assemblies which exhibit a more potent effect on the transepithelial electrical resistance of cells and transport anions through cells with a greater efficiency. Such peptides should also exhibit greater stability and a lower occurrence of multimers when added to solution. What is further needed are peptides which modulate or regulate tight junctions.